A novel soluble tissue factor variant with an altered factor VIIa binding interface.
Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.
Lee
G F
GF
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Kelley
R F
RF
D000813:Anilides / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics
D000813:Anilides; D008081:Liposomes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa